Process for removing chlorophyll present in a plant extract and extracts thus obtained

Abstract

The present invention relates to a process for removing chlorophyll present in plant extracts, in particular natural plant extracts used for cosmetic purposes and obtained by alcoholic extraction; it also relates to plant extracts with reduced chlorophyll content obtained by this process.

Description

Title of the invention: Process for removing chlorophyll present in a plant extract and extracts thus obtained

[0001] The present invention relates to a process for removing chlorophyll present in plant extracts, in particular plant extracts used for cosmetic purposes and obtained by alcoholic extraction; it also relates to plant extracts with reduced chlorophyll content obtained by this process.

[0002] Alcoholic, and in particular ethanolic, plant extracts are generally the most concentrated natural extracts in secondary metabolites. Without additional processing, these extracts, when obtained from aerial parts, contain chlorophyll.

[0003] Chlorophyll is naturally biosynthesized in plants and is essential for photosynthesis; it is also responsible for the green color of extracts obtained from plants. Regardless of the extraction solvent, traces of chlorophyll are inevitably present in extracts prepared from the aerial parts of plants (leaves in particular).

[0004] In addition to contributing to the undesirable color of the extract, chlorophyll is a photosensitizing molecule; it can also act as a pro-oxidant and thus have a detrimental impact on the activity of the extracts and therefore on the cosmetic formulas that contain them (Chotphruethipong et al., J. Food Biochem. 41, 1-10 (2017)). In order to improve the quality of plant extracts, it is preferable, even necessary, to remove the chlorophyll, in other words, to treat them by "de-chlorophyllization".

[0005] “Dechlorophyllization” is generally carried out by removing chlorophyll with activated carbon, using organic solvents, or by sedimentation (Tagrida et al., J. Food Biochem. 44, 1-14 (2020); Tagrida et al., RSC Adv. 11, 17630-17641 (2021); Olatunde et al., J. Food Biochem. 42, 1-11 (2018); Olatunde et al., Int. J. Food Sci. Technol. 56, 2804-2819 (2021)). However, the “dechlorophyllization” techniques using activated carbon and organic solvents are aggressive and remove, in addition to chlorophyll, other active molecules of interest from the plant extract. As for the sedimentation technique, it preserves more of the active molecules of the plant extract but does not allow the elimination of all the chlorophyll (the yields described in the literature are 82% according to Olatunde et al. (2020) and 73% according to Tagrida et al. (2021)).In addition, this method is lengthy and expensive; for example, the process described by Tagrida et al. (2020) includes the steps of keeping the extract cold at 4 °C for 24 hours, centrifuging at 10000 g for 30 minutes at 4 °C, collecting the supernatant, and freezing at -20 °C. then freeze-drying, its industrialization therefore requires long steps and expensive equipment (freezing and freeze-drying).

[0006] It therefore remains necessary to develop a process for de-chlorophyllizing plant extracts which allows for an efficient, economical and rapid elimination of chlorophyll while preserving the active molecules present in the plant extract.

[0007] This is what the Applicant has achieved with a new technique for purifying plant extracts which makes it possible to remove up to 98% of the chlorophyll without losing the molecules of interest.

[0008] More particularly, the invention relates to a process for dechlorophyllizing a plant extract from aerial parts of a plant in at least one non-aqueous solvent comprising the following steps:

[0009] a) adjusting the water content of said extract so that it contains at least 3%, preferably at least 10%, of water relative to the total volume of solvent in the extract;

[0010] b) evaporation of at least one non-aqueous solvent contained in the mixture obtained at the end of step a) without evaporation of the water; and

[0011] c) filtration to a cut-off threshold of at most approximately 50 µm and removal of residues.

[0012] The aerial parts of the plant classically include the leaves, stems, and flowering aerial parts. Preferably, the aerial parts of the plant consist of the leaves.

[0013] The process according to the invention can be applied to any plant extract of aerial parts of plants in at least one non-aqueous solvent; in particular, it can be obtained by mixing any dry plant extract of aerial parts of plants in at least one non-aqueous solvent. Alternatively, it can be obtained by extraction with at least one non-aqueous solvent; according to this embodiment, and by way of example and without limitation, the extract can be obtained by maceration, reflux, ultrasound, microwave extraction, etc., for example, by reflux extraction with at least one non-aqueous solvent from dried aerial parts of plants, preferably ground; and filtration or dewatering to remove solid residues.

[0014] The non-aqueous solvent is an organic solvent having a boiling point lower than that of water; it can in particular be chosen from ethanol, ethyl acetate.

[0015] The water added to the plant extract (liquid or solubilized in at least one non-aqueous solvent such as an organic solvent) in step a) is introduced in such a way that its quantity is at least 3% v / v, preferably at least 10% v / v, of water (volume of water in relation to the total volume of non-aqueous solvent and water); there is no upper limit to the quantity of water added, thus larger quantities of water may be added but it is preferable to avoid excessive quantities which will require subsequent removal of the water.

[0016] Step b) can be carried out according to conventional techniques known to those skilled in the art; in one particular embodiment, it is carried out as follows: the evaporation of the non-aqueous solvent contained in the mixture from step a) is carried out under vacuum using a Rotavopor®-type apparatus, which allows solvents to be evaporated below their boiling points using vacuum. The extract is heated to between 20 and 60 °C, preferably at 40 °C; the vacuum used is between 10 and 200 mbar, preferably at 72 mbar.

[0017] Step c) is preferably carried out with a cellulosic fiber type filtration membrane having a cut-off threshold between 0.2 and 50 pm, preferably less than 10 pm, even more preferably about 1 pm.

[0018] At the end of the process according to the invention, the bleached plant extract can be further treated by conventional steps in order to standardize it, sterilize it or stabilize it, for example by changing the final solvent, which is water and therefore not bacteriostatic, to a glycol solvent.

[0019] The process according to the invention is therefore distinguished from that of sedimentation in particular in that it does not require evaporating the extraction solvent before adding water.

[0020] In addition to its very good efficiency (total elimination of chlorophyll and preservation of the active matter in plant extracts), the process according to the invention has the advantage of:

[0021] - to be fast, it can be implemented in approximately 1 to 6 hours, in this that it involves fewer steps than the dechlorination process by sedimentation (no cooling, centrifugation and freeze-drying steps);

[0022] - to be able to be implemented with simple and inexpensive industrial equipment expensive;

[0023] - to be less expensive than carbon bleaching because it does not require to add an input other than water.

[0024] The present invention further relates to a plant extract from aerial parts of a plant which can be obtained by the process according to the invention and which is characterized in that it contains less than 0.05 mg / ml, preferably less than 0.04 mg / ml and even more preferably less than 0.03 mg / ml of chlorophyll (corresponding respectively to percentages of 0.005% w / v, 0.004% w / v and 0.003% w / v).

[0025] The present invention relates more particularly to a plant extract of aerial parts, preferably the leaves, of Combretum micranthum purified and decolorized according to the process according to the invention and characterized in that it contains less than 0.05 mg / ml, preferably less than 0.04 mg / ml and even more preferably less than 0.03 mg / ml of chlorophyll.

[0026] Combretum micranthum (kinkeliba) is a plant of the Combretaceae family. It is also known as: Bureava crotonoides; Combretum altum; Combretum flo- ribundum; Combretum parviflorum; Combretum raimbaultii; or kinkeliba (common name). It is a common plant in Africa, generally found in the Sahel countries.

[0027] Advantageously and despite the de-chlorophyllization treatment, this extract is rich in active molecules, it contains in particular between 5 and 25%, preferably between 9 and 19%, even more preferably at least 14% of kinkeloids in the dry extract; the latter may also contain flavonoids.

[0028] Such extracts of C. micranthum can be used for cosmetic purposes as a depigmenting, anti-spot, whitening, anti-redness and complexion unifying agent or for anti-aging activities, to prevent and / or treat sagging skin, loss of skin firmness, loss of elasticity of skin fibers, withered skin, thinning skin, wrinkles and fine lines and dull and / or lackluster skin. Figures

[0029] [Fig. 1] represents a diagram of an example of a process for preparing a plant extract comprising preliminary extraction steps, the steps of the process according to the invention (indicated by the letters a), b) and c)) as well as steps after the process according to the invention.

[0030] [Fig. 2] shows the UV 280 chromatograms of crude extract 1 before treatment (top) and treated extract 1 (bottom). The grey bar on the left represents the chlorophyll B peak and the one on the right, the chlorophyll A peak.

[0031] [Fig.3] represents the ELSD chromatograms of the crude extract 1 before treatment at the top and of the treated extract 1 at the bottom. Examples Preparation of extracts according to the invention

[0032] Extraction: 300 g of previously crushed Combretum micranthum leaves are treated in 2917 g of ethanol under reflux for 3 h in a 5 L flask. After 3 h of extraction, the mixture is filtered at 40 µm using an Eaton Beco Integra Lab 140 P pressure filter. This filtration yields 1915 g of extract in ethanol. The plant material is then collected for a second extraction under reflux with 2820 g of ethanol for 3 h in a 5 L flask. After 3 h, the mixture is again filtered at 40 µm. This time, the spent grains are removed. This second extraction yields 2744 g of extract in ethanol.

[0033] In total, 4659 g of crude extract are recovered at this stage. Implementation of the process according to the invention:

[0034] At this stage, 10% water is added to the total crude extract, i.e. 465 g of water added.

[0035] The organic solvent (ethanol) is then evaporated on an evaporation system under A Buchi "Rotavapor R-300" vacuum evaporator is used. Ethanol is evaporated at 40°C and 100 mbar. However, care must be taken not to evaporate the added water.

[0036] The mixture is then filtered at 1 µm.

[0037] Following this filtration, the treated extract, i.e. decolorized and purified, of Combretum micranthum is obtained. Analysis of several plant extracts

[0038] In order to evaluate the yield of the process according to the invention, the determination of chlorophylls A and B (according to Olatunde et al., 2018 and AO AC (2002) Official Method of Analysis) was carried out on different extracts:

[0039] - those prepared as described above without treatment to remove the chlorine Rophyllum (crude extract); and

[0040] - those prepared as described above with treatment to eliminate the chlorine rophylle (treated extract).

[0041] [Tables 1] Samples Chlorophyll A (mg / ml) Chlorophyll B (mg / ml) Total Chlorophyll (mg / ml) Reduction of total chlorophyll from crude Crude Extract 1 1.02 0.72 1.74 Treated Extract 1 0.01 0.03 0.04 43.5 Crude Extract 2 0.99 0.75 1.74 Treated Extract 2 0.02 0.03 0.05 34.8 Crude Extract 3 1.05 0.68 1.73 Treated Extract 3 0.01 0.02 0.03 57.7

[0042] Table 1: Table of chlorophyll dosage in 3 extracts from the same plant

[0043] These results show an excellent efficiency of chlorophyll A and B removal by the process according to the invention; these yields are much higher than those described in the literature.

[0044] In addition, the chromatographic profiles of the crude and treated extracts were compared to ensure that the loss of active material is reduced; the chromatograms in Figures 2 and 3 confirm the elimination of chlorophyll (right peaks) and the preservation of the molecules of interest (left peaks).

Claims

Demands

1. A process for dechlorophyllizing a plant extract from aerial parts of a plant obtained by extraction with a non-aqueous organic solvent having a boiling point lower than that of water, comprising the following steps: a) adjusting the water content of said extract so that it contains at least 3%, preferably at least 10%, of water relative to the total volume of solvent contained in the extract; b) evaporating at least one non-aqueous solvent contained in the mixture obtained at the end of step a) without evaporating the water; c) filtering to a cut-off of at most about 50 µm and removing the residues.

2. A dechlorophyllization process according to claim 1, characterized in that step b) is carried out by evaporation under vacuum with heating of the mixture obtained at the end of step a) to a temperature between 20 and 60 °C and with a vacuum between 10 and 200 mbars.

3. 3. Dechlorophyllization process according to claim 1 or claim 2, characterized in that the filtration of step c) is carried out at a cut-off threshold of less than 10 pm.

4. 4. Dechlorophyllization process according to any one of claims 1 to 3, characterized in that said extract is obtained from aerial parts of Combretum micranthum.

5. 5. Plant extract of aerial parts of plant obtained by the process according to any one of claims 1 to 4, characterized in that it contains less than 0.05 mg / ml of chlorophyll.

6. 6. Plant extract of aerial parts of Combretum micranthum capable of being obtained by the process according to any one of claims 1 to 4, characterized in that it contains less than 0.05 mg / ml of chlorophyll.

7. 7. Plant extract of aerial parts of Combretum micranthum according to claim 6, characterized in that it contains between 5 and 25%, preferably between 9 and 19%, more preferably at least 14% of kinkeloids in the dry extract.

8. 8. Cosmetic use of a plant extract of aerial parts of Combretum micranthum according to claim 6 or claim 7, as a depigmenting, anti-spot, whitening, anti-redness and even- complexion corrector or for anti-aging activities, to prevent and / or treat sagging skin, loss of skin firmness, loss of elasticity of skin fibers, withered skin, thinning skin, wrinkles and fine lines and dull and / or lackluster skin.

Images